Phase Behavior Of Membrane Lipids Research Articles

Combination of MD Simulations with Two-State Kinetic Rate Modeling Elucidates the Chain Melting Transition of Phospholipid Bilayers for Different Hydration Levels.

The phase behavior of membrane lipids plays an important role in the formation of functional domains in biological membranes and crucially affects molecular transport through lipid layers, for instance, in the skin. We investigate the thermotropic chain melting transition from the ordered Lβ phase to the disordered Lα phase in membranes composed of dipalmitoylphosphatidylcholine (DPPC) by atomistic molecular dynamics simulations in which the membranes are subject to variable heating rates. We find that the transition is initiated by a localized nucleus and followed by the propagation of the phase boundary. A two-state kinetic rate model allows characterizing the transition state in terms of thermodynamic quantities such as transition state enthalpy and entropy. The extrapolated equilibrium melting temperature increases with reduced membrane hydration and thus in tendency reproduces the experimentally observed dependence on dehydrating osmotic stress.

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (ld) domains on the surface of GUVs composed of 40:60mol% DOPC/DPPC, over a wide range of temperatures (14–25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of LissamineTM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.

Membrane biophysics

Membrane biophysics

Structure and function of membrane rafts

Abstract Lipids do not always mix uniformly in membranes, but can cluster to form microdomains. We will consider one type of microdomain that can form in cell membranes. These are enriched in cholesterol and sphingolipids, and are referred to as rafts. Rafts probably exist in membranes in the liquid-ordered phase or a phase with similar properties. We will briefly review membrane lipid phase behavior, and the differences between liquid-crystalline, liquid-ordered, and gel-phase membrane bilayer domains. We will present evidence suggesting that phospholipid-rich, liquid-crystalline phase domains and sphingolipid-rich, liquid-ordered phase domains (rafts) can exist in equilibrium in biological membranes, especially the plasma membrane. Preferential partitioning of membrane proteins into rafts can affect function. Among the proteins that are targeted to rafts are those anchored in the outer leaflet of the membrane through covalent attachment to a special glycolipid, glycosyl phosphatidylinositol (GPI). Other proteins that are linked to saturated acyl chains, such as those that are directly acylated with two or more palmitate chains, or a palmitate and a myristate chain, are also targeted to rafts. Targeting of GPI-anchored proteins and other proteins to rafts plays a role in signal transduction in hematopoietic cells, and possibly also in sorting in intracellular membranes and regulation of cell-surface proteolysis in other mammalian cells.

Thermal acclimation of phase behavior in plasma membrane lipids of rainbow trout hepatocytes.

The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl] dipalmitoyl-L-alpha-phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20 degreesC) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20 degrees C (13.2 +/- 0.7 degrees C)- than 5 degrees C (7.2 +/- 0.1 degrees C)-acclimated trout. Similarly, acclimation from 5 to 20 degrees C increased both the onset temperature (from 2.0 +/- 0.3 to 7.2 +/- 0.6 degrees C) and the thermal range (from 10.9 +/- 0.5 to 16.0 +/- 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately -2 degrees C) and completion temperatures (-9 degrees C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58 degrees C; only membranes of 5 degrees C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 +/- 1.7 degrees C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.

Phase behaviour of membrane lipids containing polyenoic acyl chains

The low-temperature thermal behaviour of di-18:2 phosphatidylethanolamine (di-18:2 PE) is shown to be characterised by similar broad low-enthalpy transitions to those previously reported for polyenoic samples of phosphatidylcholines (Keough and Kariel (1987) Biochim. Biophys. Acta 902, 11–18), and monogalactosyldiacylglycerol (Sanderson and Williams (1992) Biochim. Biophys. Acta. 1107, 77–85). Real-time X-ray diffraction measurements indicate that these transitions correspond to transitions between the gel (L β) and liquid-crystal (L α) phases of the lipids. The gel phase of these lipids is, however, much more loosely packed than the corresponding phases of membrane lipids containing monoenoic or fully-saturated acyl chains. The low enthalpy and reduced co-operativity of the L α a ̊ L β phase transitions of the polyenoic lipids is attributed to the reduced contribution of van der Waals interactions between their acyl chains in the gel-state of these lipids. Comparison with the earlier results obtained for MGDG suggest that the acyl chains of polyenoic lipids can form well-ordered lattices but require the additional energy input associated with the formation of a hydrogen bond network between the lipid headgroups in order to do so.

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.

Low-temperature phase behaviour of the major plant leaf lipid monogalactosyldiacylglycerol

Heating and cooling thermograms of unsaturated MGDG samples isolated from the leaves of Vicia faba arc suprisingly featureless. This reflects the low enthalpies associated with phase transitions in highly unsaturated lipids and the fact that these transitions, in the case of MGDG, are to a large extent masked by those associated with the freezing and melting of ice. Careful choice of thermal heating/cooling regimes, combined with the use of real-time X-ray diffraction and freeze-fracture measurements, permits a detailed analysis of the phase behaviour of the system. The phase behaviour of unsaturated MGDG sample is shown to be basically similar to that seen in saturated MGDG samples. The lipid which exists in the inverted hexagonal (Hex II) liquid crystal phase at room temperature forms a highly disordered lamellar gel (L β) phase on cooling to temperatures below about −15°C. On reheating, this first reorganises at a temperature of about −10°C to form a well-defined L cl phase. Above about −2°C, this melts to re-form the Hex II phase. Samples re-cooled from temperatures between −2°C and 14°C revert directly to the L cl phase while samples cooled from higher temperatures form the L β phase. This reflects the fact that the former samples contain small amounts of unmelted L cl phase lipid. The implications of these observations are discussed in terms of the general problems associated with the measurement of low-temperature phase behaviour of membrane lipids.

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.

Membrane lipid phase behavior and lipid-protein interactions.

One of the most distinguishing features of nearly all biological membranes is that they contain a highly complex assortment of polar lipids. Only a few major lipid classes can be recognized but there is a whole spectrum of molecular species within each of these major lipid classes which differ in the type, length, and number of unsaturated residues of their hydrophobic component. There is a general consensus, based on observations that have been obtained by a variety of biophysical methods, that the lipid constituents of all biological membranes are arranged in a bilayer configuration in which the polar groups are located on the outside, in contact with the aqueous medium, and the hydrocarbon substituents are oriented toward the interior to form a hydrophobic domain that excludes water. It has been argued on the basis of the hydrophilic to hydrophobic balance within the molecules that membrane lipids have a relatively low critical micellar concentration and a discrete distribution of domains within the molecule and this is responsible for creating a stable bilayer structure.KeywordsAcyl ChainHydrocarbon ChainAcyl Chain LengthBilayer PhaseLateral Phase SeparationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The combined and separate effects of temperature and freezing on membrane lipid phase behavior: Relevance to cryobiology

The combined and separate effects of temperature and freezing on membrane lipid phase behavior: Relevance to cryobiology

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the nonlamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to nonlamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T m or headgroup mobility (monitored by 2H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.